Biosulphidogenesis and Bioaccumulation of sulphate by

Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502
Vol. 3(ISC-2013), 203-208 (2014)
Res. J. Recent. Sci.
Biosulphidogenesis and Bioaccumulation of sulphate by moderately
Thermophilic, Facultative anaerobic Bacteria Aeromonashydrophilaisolated
from hot Water spring
Patil S. Z., Unnitha. A. R. and Unnikrishnan G.
Department of Biotechnology, Birla College of Arts, Science and Commerce, Kalyan, Maharastra, INDIA
Available online at: www.isca.in, www.isca.me
Received 9th January 2014, revised 23rd February 2014, accepted 28th March 2014
Abstract
A unique, facultative anaerobic, moderately thermophilic Sulphate reducing prokaryote (TSRP) was isolated from
Vajreshwari and Ganeshpuri hot Springs of Thane, Maharashtra. The optimum temperature, pH and NaCl concentration for
growth of this strain was found to 41°C, 6.5, and 4.5% respectively. The strain Si showed 100% reduction
ofstandardsulphatein 11hrs., with no production of sulfide. This result reflects the presence of assimilatory sulfate reduction
pathway type I, which reduces sulfate to sulfite and finally to sulfide that is accumulated in the cellfor cysteine biosynthesis.
The analysis of the effluent collected from colour and dye industry showed high concentration of sulphate (689ppm).These
effluent wassubjected to sulphidogenesis by strain Si and there was complete reduction of sulphate in 12.30 hrs.with no
production of sulphide. Phylogenetic analysis of 16s rRNA sequence placed strain Si in gamma subclass of proteobacter,
showing highly similarity with other phylogenetic relatives. Thus this isolate is a member of genus Aeromonas and the type
strain is hydrophila strain ZHYYZ-1. The result indicated that Aeromonashydrophila strain ZHYYZ-1 has high efficiency of
sulphate reduction in much less time with no production of sulfide than previously studied anaerobic bacteria.
Keywords: Biosulphidogenesis, TSRP, Assimilatory Sulphate reduction pathway Type I, Bioaccumulation, 16s rRNA.
Introduction
Sulphates are occur naturally occurring element and are used in
the manufacture of chemicals, dyes and fertilizers, in the mining
and pulping industries, in sewage treatment and in wood
preservation1.
Sulphates are discharged into the aquatic environment through
effluents from industries like mining, smelting, pulp and paper
mills. Surface water can also be contaminated by atmospheric
sulphur dioxide2.
Sulphate is one of the least toxic anions. The major
physiological effects resulting from ingesting large quantities of
Sulphate are catharsis and gastrointestinal irritation. These
effects are enhanced when Sulphate is consumed in combination
with magnesium1. The maximum permissible limit for Sulphate
in water is 500ppm (WHO). Thus, biosulphidogenesis (Sulfide
or elemental Sulfur generation) is necessary before discharging
the Sulphate containing effluent into water body. Since the hot
springs contain a wide range of sulphate concentration,
thermophile microorganisms are being used for sulphidogenesis
nowadays. The reduction of sulphate by this process is
environment friendly and economically viable hence they are
being widely used in bioremediation process3.
Thermophilic sulfate-reducing prokaryotes (TSRP) have
increasingly attracted interest due to their potential in various
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biotechnological applications, such as biosulphidogenesis and
biohydrometallurgical processes4.
One of the best applications of TSRP is treating the effluent
having sulphateconcentration above the permissible limit. The
effluent from Textile, Battery, Paper andpulp, Colour and dye
industry contain high amount of sulphate. These runoff
ultimately affects the quality of the river or water body beside
these industries and effects environment, wildlife as well as
human. Hence, it is advisable to treat these effluent for reducing
sulphate by TSRP.
The objective of the present study is to enrich, isolate and
characterize TSRP from a Vajreshwari and Ganeshpuri hot
springs stretching about 7 km in the bed of the River Tansa,
Thane, Maharastra. (http:// www.mumbaisuburbs.com /
mumbai-tourist
/
travelvajreshwari.html)
and
study
biosulphidogenesis of industrial effluent containing high amount
of sulphate.
Material and Methods
Sampling: Water samples were collected from seven different
hot springs (from Vajreshwariand Ganeshpuri, Thane) Mumbai.
Surface water samples were taken from the Hot Springs using a
grab sampler. Sample was stored in clean polyethylene
container with lid. The temperature of the sample was taken
with a laboratory thermometer and recorded. All samples were
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Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502
Vol. 3(ISC-2013), 203-208 (2014)
Res. J. Recent. Sci.
taken on the same day to prevent discrepancies due to sample
date. Water sample used for inorganic analyses was
immediately fixed with 3 ml of a 20% (wt/vol) zinc acetate
solution for sulfate analysis andwith the same volume of a
90mM zinc acetate solution for determination of the sulfide
concentration. In situ spring water used in the test for sulfide
production by TSRP was kept in a sterile plastic bottle
(1,000ml) 3.
Media and growth Conditions: Medium 77 described by
Postgate was used for routine stock maintenance and all
enrichment culture studies5. Medium 77 contained (g/ Lit.:
K2HP04, 0.5; NH4C1, 1.0; CaC12,.2H20, 0.1; MgS04, 7H2,0,
0.1; sodium lactate, 5; yeast extract, 1.0; FeS04,.7H20, 5 ;
sodium thioglycolate, 1.0; and ascorbic acid, 1.0). Black
colonies were isolated from Medium 77 and 4% (w/v) purified
agar. Stock cultures of all strains were prepared from single
isolated colonies that proliferated on transfer in Medium 77. All
stock cultures were incubated at 50°C.
Characterization and identification of the isolates:
Morphological Studies: Morphological properties were
investigated by using 18 hour old bacterial cultures. These
included the wet mount preparations using light microscope and
Gram staining to confirm Gram reaction. Motility was
determined by hanging drop method6.
Biochemical Tests: The thermophilic isolate was identified by
presumptive conventional, physiological and biochemical tests.
These tests were; Gram reaction, catalase production, hydrolysis
of protein, starch and lipid, and acid production from sugar6.
The species was reconfirmed in an automated Biomerieux Vitek
2 System (At Nucleus Diagnostic Centre, Kalyan).
Optimization of Growth Conditions: Determination of the
Optimum pH: The optimum pH for growth was determined by
using phosphate buffer, universal buffer, and Tris –HCI buffer
to obtain different pH values in the range of 4.0 to 9.0 pH and
was confirmed using pH meter6.
biosulphidogenesis. The sites for sample collection were within
the city zone of Thane. Samples were collected in polyethylene
bottles. The sample was first analysed to find out the
concentration of sulphate (Turbidometric method, APHA).
Effluent were then exposed to TSRP for reduction of sulphate to
sulphide.
Biosulphidogenesis: Sulfate reduction rate (SRR):
Measurement of the reduction of sulphate into sulphide was
performed method described in APHA. The rate of sulphate
reduction was determined from points taken during a 10 hr time
course. All assays were done in triplet.
Sulfide production rate: Dissolved sulfide concentrations were
estimated by using Iodometric method (APHA) after in situ
fixation with a zinc acetate solution. Concentration of sulfide
was detemined both before and after process of sulphate
reduction to estimate actual amount produced by
biosulphidogenesis. All assays were done in triplet.
Strain identification by 16s rRNA Analysis: The isolated
colony was sequenced for its conserved sequences and analysed
for partial 16s rRNA (Sequencing was done at gene Ombio,
Pune, Maharashtra).
The predicted 16S rRNA sequences from this study were
compared with 16S rRNA sequences in database available in
ribosomal database project (release 8.1;http:// rdp8.
cme.msu.edu.). Comparisons were made using the program
BLASt (ftp://ftp.ncbi.nih.gov/ BLAST/ executables/LATEST/)4.
Phylogenetic and Evolutionary Analysis: For cladogram
construction, partial 16S rRNA gene sequences representing the
15 most prevalent OTUs (Operational Taxonomic Units) from
thermophilic environment (NCBI database) were aligned using
CLUSTALW. Phylogenetic and molecular evolutionary
analyses were performed using software cladogram.
Results and Discussion
Determination of the Optimum Temperature: Cultures were
streaked onto agar plates and incubated at a range of
temperatures from 20-60°C. The plates were observed daily up
to 5 days7.
Characterization of in situ sulphidogenesis: Microbial sulphate
reduction at high temperatures was studied using samples
collected from several sites in Vajreshwariand Ganeshpuri hot
springs. Enrichment cultures were initiated with Medium 77.
After incubation of 24 hrs.dense black coloured colonies were
transferred to fresh media for identification.
Growth at Different Sodium Chloride Concentration: The
experiments were carried out containing 100 ml of isolation
medium prepared in a phosphate buffer at final concentration of
50mM and at a final salt (NaCl) concentration of 0.5%, 1.5%,
3.0%, 4.5%, 6.0% and 7.5%,. 1 ml of culture was added and the
flask was incubated at 41°C in an orbital shaker running at 200
rpm. The growth was determined at 3h intervals by measuring
the O.D at 550 nm5.
Sample Collection of effluent from industries: The effluent
from colour and dye industry wasselected as sample for
Cellular properties: Cells appeared as very tiny straight rods.
Motility was not observed. Exponential phase cells stained
Gram-negative and lacked catalase. The optimum peak
concentration for growth was at 4.5% NaCl. Other biochemical
properties (As per Bergey’s manual) are described in table 1.
Species was again confirmed by using automated system of
Biomerieux System (Tested at Nucleus diagnostic Centre,
Kalyan) stated as in table-2.
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Vol. 3(ISC-2013), 203-208 (2014)
Res. J. Recent. Sci.
Table-1
Biochemical Properties of TSRP (According to Bergey’s manual)
Characteristics
Strain si
Gram Nature
Gram Negative
Shape
Curved
Motility
M
Temperature°C – Optimum
41
pH - Optimum
6.5
NaCl % - Optimum
4.5
Oxidase
Catalase
Casein
+
Starch
+
D-Glucose
+
D-Fructose
+
Maltose
+
Mannose
Trehalose
Cellobiose
Sucrose
Mannitol
Melibiose
+
Lactose
+
Arabinose
Xylose
Galactose
+
Nitrate
Citrate
Key: Growth : + ; No Growth : - ; M : Motile
2
APPA
-
3
ADO
10
H2S
-
11
BNAG
17
BGLU
-
18
23
ProA
-
26
33
SAC
-
34
40
ILATk
-
46
GlyA
58
0129R
Table-2
Biochemical Tests (By Biomerieux Vitek 2 System)
Biochemical Details
- 4
PyrA
- 5
IARL
7
d CEL
-
9
BGAL
-
-
12
AGLTp
-
13
d GLU
+
14
GGT
-
15
OFF
dMAL
+
19
dMAN
-
20
dMNE
-
21
BXYL
-
22
BAlap
-
LIP
-
27
PLE
-
29
TyrA
-
31
URE
-
32
dSOR
-
d TAG
-
35
d TRE
-
36
CIT
-
37
MNT
-
39
5KG
-
41
AGLU
-
42
SUCT
-
43
NAGA
-
44
AGAL
-
45
PHOS
-
-
47
ODC
-
48
LDC
-
53
IHISa
-
56
CMT
+
57
BGUR
-
-
59
GGAA
-
61
IMLTa
-
62
ELLM
-
64
ILATa
-
Growth and metabolic properties: Long lag period was not
observed and adoubling time of less than 4 hr was observed
during the exponential growth phase, figure 1.
Bacterium had an optimal growth temperature near 41oC; it
grew below 65° C and above 37°C, figure2.
Themicrobe showed growth in between pH range of 6.0 to 8.0
in Medium 77 with an optimal pH near 6.5.and optimum growth
was found at NaCl concentration of 4.5%.
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Standard sulphate Reduction: 100 % reduction was observed
for a standard sulphate from500 mg/L (ppm) to 0 mg/L (ppm) in
11 hrs without the production of sulfide, table 3.
Effluent treatment: The effluent from colour and dye industry
contained high concentration of sulphate well above the
permissible limit (500 ppm). The time taken by TSRP for
complete reduction of sulphate was found to be 12.30hrs.with
no production of sulphide, table3.
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Res. J. Recent. Sci.
16s rRNAAnalysis : The sequence of conserved sites by partial
sequence analysis gave the following sequences.
TCGCAATTGGCGGGCGGTCTACACATGCAAGTCGAGC
GGCAGCGGGGAAAGTAGCTTGCTACTTTTGCCGGCGA
GCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCA
GTCGAGGGGGATAACAGTTGGAAACGACTGCTAATAC
CGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGG
GCCTTGCGCGATTGGATATGCCCAGGTGGGATTAGCTA
GTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCT
AGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTG
AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGG
GAATATTGCACAATGGGGGAAACCCTGATGCAGCCAT
GCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCA
CTTTCAGCGAGGAGGAAAGGTTGATGCCTAATACGTA
TCAACTGTGACGTTACTCGCAGAAGAAGCACCGGCTA
ACTCCGTG
20 30 40 50 60
Temperature (O C)
Figure-2
Optimum temperature and growth yield of Strain Si. The
organism was cultured in Medium 77 at the temperatures
indicated
The sequence was aligned for comparison with the existing
databases by using in silico tool BLAST. The isolate was found
to be of genus Aeromonas and type strain was found to be
hydrophila strain ZHYYZ-1.
Figure-1
Growth curve of Strain Si cultured on sulphate in modified
Medium 77
The phylogenetic and evolutionary analysis of the operational
taxonomic units isolated from thermophilic environment was
analysed by CLUSTALW, figure 3 and it showed the following
relationship between the different species. The score table
showed the % similarity of these strain and all other OTUs,
Table 4.
Table-3
Initial and final concentration of sulphate and sulphide before and after the treatment of effluent collected from 3 industries
by TSRP strain Si.
Sulphate
Sulphide
Industry /
Sulphide
Sulphate concentration (mg/L)
Reduction rate
Production rate
Standard
concentration (mg/L)
(%)
(%)
Standard Sulphate
Colour and dye
Industry
Reduction
Initial
Final
500
0
11
689
0
12.30
Initial
Final
100
--
--
0
100
12
12
0
Time ( hrs.)
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Table-4
The Score Table: Percent Similarity Index (As per Sequence Similarity)
Accession number of Query Organism - >gi|290792568|gb|GU563992.1| Aeromonashydrophila strain ZHYYZ-1
Accession Number of OTUs
Score
>gi|44194255|gb|AY538658.1| Aeromonashydrophila strain HY03
96.69
>gi|238814979|gb|FJ972531.1| Pseudomonas aeruginosa
85.06
>gi|82941283|dbj|AB242868.1| Marinomonasostreistagni
84.86
>gi|188219225|emb|AM999769.1| Thermusaquaticus
76.60
>gi|143692855|gb|EF426770.1| Geobacillus sp. DDS021
76.53
>gi|219857149|ref|NR_024777.1|Thermanaeromonas toyohensis strain ToBE
76.16
>gi|51036225|dbj|AB186359.1| Clostridium clariflavum
75.68
>gi|440576572|emb|HF558369.1| Thermus thermophiles
75.68
>gi|540352423|emb|HG380021.1| Streptococcus thermophiles
74.47
>gi|576620|gb|L37731.1|VIBRR16SAG Vibrio anguillarum
73.75
>gi|378405442|gb|JQ346745.1| Thermodesulfobacterium commune YSRA-1
73.35
>gi|35210323|dbj|AB089844.1| Sulfobacillusthermosulfidooxidans
73.08
>gi|61653285|dbj|D28576.2| Sphingomonasmali
63.33
>gi|44735|emb|X00084.1| Methanococcusvanniellii
49.66
Figure-3
Cladogram
Discussion: The present study intend to identify the
assimilatory sulphate reduction in thermophilic bacteria8.
Bacterial sulphate reduction at high temperatures appears wide
spread in Vajreshwari and Ganeshpuri hot springs and was
associated with at leastone species. Thermophilic sulphatereducing bacteria were found in hot water spring where sulphate
content
and
organic
matter
content
are
more.
Biosulphidogenesis appeared most active in the sulphatedepleted thermal ecosystem, where microbial sulfate reduction
occurred.
production as high as 150 mg/L. This showed that as compared
with anaerobic sulphate reduction, the reduction rate by aerobic
TSRP is faster and more effective as there is no production of
sulfide. The reason behind this is that the facultative aerobes
require the sulphide for synthesis of other biomolecule like
cysteine (Biocyc database). Thus sulphate is reduced to sulfide
first and then it is consumed for their nutrient requirement. This
indicates that the rate of sulphate reduction seems to be high in
this strain as compared to anaerobic organism (where
dissimilatory reduction is present)9.
The isolated strain Si was found to be facultative anaerobic,
moderate thermophilic, Gram negative rods. Optimum
temperature for growth was 41°C. pH range 6.0-8.0. Growth
inhibited by 4.5% NaC1.
The analysis of the effluent collected from Colour and dye
industry showed sulphate level above the permissible limit (500
ppm)10. These effluent was subjected for sulphidogenesis by
TSRP strain Si, and the reduction rate was found to be 100% in
12.30 hrs.withno production of sulphide. It takes more time for
reduction of the effluent sample,may be because of presence of
other elements like H2S, Cd, Ni, Cu, Cd, Cr, Pb (Reis MA,
Almeida JS, Lemos PC, Carrondo MJ., 1992 and Aili Tan,
Kaixuan Tan, Zhengji Yi 2004) that slow downs the rate of
sulphate reduction.
Strain Si showed complete reduction of standard sulphate
(500ppm to 0 ppm) in 11hr with no production ofsulphide
production. Experiments by J. Suschka and L. Przywara(2006)
showedthat 90 % sulphate reduction was achieved after 60hrs.
by using Desulfovibrio or Desulfobacterium with sulfide
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Vol. 3(ISC-2013), 203-208 (2014)
Res. J. Recent. Sci.
Biochemical tests (Both Manual and automated method) and
16s rRNA analysis confirms that species of reference is of
Genus Aeromonas and species as hydrophila. strain ZHYYZ-1.
The phylogenetic analysis and percent similarity
indexnshowed that A. hydrophilastrainZHYYZ-1have
96.69% similarity with its closely related species of A.
hydrophila strain HYO.
The score table showed that sequences in study has 85.06%,
84.86% similarity with Pseudomonas aeruginosaand
Marinomonasostreistagnirespectively. The evolutionary tree
showed relationship between all the 15 species. It was found out
that A. hydrophilastrain ZHYYZ-1, A. hydrophilaHYO
strain,Pseudomonasaeruginosaand Marinomonasostreistagniare
evolved from common ancesters.
The study showed that A. hydrophila strain ZHYYZ-1 being a
facultative anaerobe, does not require any specific bioreactor as
thtat are needed for anaerobic organism. The most important
factor is that have high potential of sulphate reduction with no
production of sulphide, thus there is no foul odour and even no
precipitation of other metal present in the effluent. Considering
all these aspects A. hydrophilastrainZHYYZ-1 will be an ideal
candidate for biosulphidogenesis and will be best way for the
treatment of industrial effluent containing high level of sulphate
Journal of Scientific and Engineering Research, 4(11)
(2013)
3.
Vemula M., Ambavaram V., Kalluru G. and Gajulapalle
M., Tollamadugu N. An Overview on Research Trends in
Remediation of Chromium, Research Journal of Recent
Sciences, 2(1), 71-83, January (2013)
4.
Anna H. Kaksonen, Jason J. Plumb,Wendy J. Robertson,
Stefan Spring, Peter Schumann, Peter D. Franzmann and
Jaakko A. Puhakka, Novel Thermophilic Sulfate-Reducing
Bacteria From A Geothermally Active Underground Mine
In Japan. Appl Environ Microbiol., May, 72(5), 3759–
3762 (2006)
5.
Carine Audiffrin, Jean-Luc Cayol, Catherine Joulian,
Laurence Casalot, Pierre Thomas, Jean-Louis Garcia and
Bernard Ollivier, Desulfonauticussubmarinus gen. nov., sp.
nov., a novel sulfate-reducing bacterium isolated from a
deep-sea hydrothermal vent, International Journal Of
Systematic and Evolutionary Microbiology, (5), 1585-1590
(2003)
6.
Francis Amala Rejula and Masilamai Dhinakaran,
Removal of Zinc (II) by Non Living Biomass of Agaricus
Bisporus, Research Journal of Recent Sciences, 1(9), 1317 (2012)
7.
Elisa
Bayraktarov, Roy
E.
Price, Timothy
G.
Ferdelman and Kai Finster, The pH and pCO2 dependence
of sulfate reduction in shallow-sea hydrothermal CO2 –
venting sediments (Milos Island, Greece), Front. In Micro
(2013)
8.
Mehar Fatma, M. Iqbal R., Khan Asim Massod and nafees
A. Khan, Coordinate changes in assimilatory Sulphate
reduction are correlated to salt tolerance : Involvemetn of
Phytohormones, Annual review and Research in biology,
3(3) (2013)
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Z. Manafi1, M. Hashemi1, H. Abdollahi,Gregory. J.
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Conclusion
The
isolated
moderarely
thermophilic
organism
Aeromonashydrophila. strain ZHYYZ-1 was Gram negative
rods with facultative anaerobic strain which has higher sulphate
reduction rate than anaerobic TSRP with no production of
sulfide.
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